Limits...
Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

Mahadevaiah SK, Bourc'his D, de Rooij DG, Bestor TH, Turner JM, Burgoyne PS - J. Cell Biol. (2008)

Bottom Line: In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response.We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs.Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11- mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

Show MeSH

Related in: MedlinePlus

In Dnmt3l- pachytene spermatocytes, increasing levels of asynapsis attenuate the MSUC response. (a and b) A rare pachytene spermatocyte with asynapsis restricted to what are probably the X and Y axes. ATR has been recruited to the asynapsed axes and has spread to the associated chromatin, and there is phosphorylation of H2AX throughout the associated chromatin. (c–f) With increasing asynapsis, the ATR becomes more focal and axially restricted, and the γH2AX staining becomes weaker and progressively more fragmented. Arrows indicate axes with partner exchange indicative of nonhomologous synapsis. (g and h) Staining of a spermatogenic cell squash preparation for the midpachytene marker H1t and for γH2AX shows that the majority of H1t-positive cells have fragmented γH2AX staining; only rare cells have a single sex body–like γH2AX-positive domain (bottom insets). In the control (top insets), cells with fragmented γH2AX staining (presumed to be zygotene) are H1t negative, whereas all H1t-positive spermatocytes have a single γH2AX-positive sex body (the small H1t-positive cells are round spermatids). (i) Quantitation of the whole nuclear γH2AX signal for rare pachytene spermatocytes with only the X and Y axes asynapsed and for pachytene spermatocytes with increasing levels of asynapsis shows that the amount of γH2AX does not increase in response to asynapsis (fitted blue regression line). The projected increase in γH2AX signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. Bars: (a–f) 10 μm; (g and h) 15 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2483523&req=5

fig3: In Dnmt3l- pachytene spermatocytes, increasing levels of asynapsis attenuate the MSUC response. (a and b) A rare pachytene spermatocyte with asynapsis restricted to what are probably the X and Y axes. ATR has been recruited to the asynapsed axes and has spread to the associated chromatin, and there is phosphorylation of H2AX throughout the associated chromatin. (c–f) With increasing asynapsis, the ATR becomes more focal and axially restricted, and the γH2AX staining becomes weaker and progressively more fragmented. Arrows indicate axes with partner exchange indicative of nonhomologous synapsis. (g and h) Staining of a spermatogenic cell squash preparation for the midpachytene marker H1t and for γH2AX shows that the majority of H1t-positive cells have fragmented γH2AX staining; only rare cells have a single sex body–like γH2AX-positive domain (bottom insets). In the control (top insets), cells with fragmented γH2AX staining (presumed to be zygotene) are H1t negative, whereas all H1t-positive spermatocytes have a single γH2AX-positive sex body (the small H1t-positive cells are round spermatids). (i) Quantitation of the whole nuclear γH2AX signal for rare pachytene spermatocytes with only the X and Y axes asynapsed and for pachytene spermatocytes with increasing levels of asynapsis shows that the amount of γH2AX does not increase in response to asynapsis (fitted blue regression line). The projected increase in γH2AX signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. Bars: (a–f) 10 μm; (g and h) 15 μm.

Mentions: Combined SYCP3, γH2AX, and ATR immunostaining indicated that in pachytene cells with limited asynapsis, there was a robust MSUC response covering the unsynapsed chromosome regions, including the unsynapsed X and Y axes (Fig. 3, a and b). However, with increasing asynapsis, the unsynapsed chromatin–located γH2AX staining became progressively more fragmented and weaker, and this was associated with increasingly focal ATR staining that was axially restricted without the spreading to adjacent chromatin that characterizes the MSUC response (Fig. 3, c–f). This fragmented γH2AX staining resembled that of meiotic DSB-associated γH2AX (Mahadevaiah et al., 2001). Amounts of γH2AX appeared to remain relatively constant in the face of increasing asynapsis; this was confirmed by quantitating the γH2AX signal (Fig. 3 i). Because the cells with fragmented and weak γH2AX staining had multiple regions of asynapsis and lacked a sex body or pseudo–sex body, we were concerned that they might have been zygotene spermatocytes, although there was often nonhomologous synapsis with the more extensive asynapsis (Fig. 3, e and f), which we consider a robust pachytene marker. For further confirmation, we stained testis tubule squashes for γH2AX and for the midpachytene marker histone H1t (Moens et al., 1997; Inselman et al., 2003), which appears during epithelial stage IV (Fig. S5, a–c; available at http://www.jcb.org/cgi/content/full/jcb.200710195/DC1). This confirmed that there were abundant H1t-positive Dnmt3l−/− spermatocytes that had fragmented γH2AX staining and no sex body; only rare midpachytene cells with a single sex body–like domain were seen (Fig. 3, g and h). Previously, Bourc'his and Bestor (2004) failed to detect H1t-positive Dnmt3l−/− spermatocytes with the same antibody, but they used spread cell preparations in which we have found that the weak stage IV H1t staining is not apparent.


Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

Mahadevaiah SK, Bourc'his D, de Rooij DG, Bestor TH, Turner JM, Burgoyne PS - J. Cell Biol. (2008)

In Dnmt3l- pachytene spermatocytes, increasing levels of asynapsis attenuate the MSUC response. (a and b) A rare pachytene spermatocyte with asynapsis restricted to what are probably the X and Y axes. ATR has been recruited to the asynapsed axes and has spread to the associated chromatin, and there is phosphorylation of H2AX throughout the associated chromatin. (c–f) With increasing asynapsis, the ATR becomes more focal and axially restricted, and the γH2AX staining becomes weaker and progressively more fragmented. Arrows indicate axes with partner exchange indicative of nonhomologous synapsis. (g and h) Staining of a spermatogenic cell squash preparation for the midpachytene marker H1t and for γH2AX shows that the majority of H1t-positive cells have fragmented γH2AX staining; only rare cells have a single sex body–like γH2AX-positive domain (bottom insets). In the control (top insets), cells with fragmented γH2AX staining (presumed to be zygotene) are H1t negative, whereas all H1t-positive spermatocytes have a single γH2AX-positive sex body (the small H1t-positive cells are round spermatids). (i) Quantitation of the whole nuclear γH2AX signal for rare pachytene spermatocytes with only the X and Y axes asynapsed and for pachytene spermatocytes with increasing levels of asynapsis shows that the amount of γH2AX does not increase in response to asynapsis (fitted blue regression line). The projected increase in γH2AX signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. Bars: (a–f) 10 μm; (g and h) 15 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483523&req=5

fig3: In Dnmt3l- pachytene spermatocytes, increasing levels of asynapsis attenuate the MSUC response. (a and b) A rare pachytene spermatocyte with asynapsis restricted to what are probably the X and Y axes. ATR has been recruited to the asynapsed axes and has spread to the associated chromatin, and there is phosphorylation of H2AX throughout the associated chromatin. (c–f) With increasing asynapsis, the ATR becomes more focal and axially restricted, and the γH2AX staining becomes weaker and progressively more fragmented. Arrows indicate axes with partner exchange indicative of nonhomologous synapsis. (g and h) Staining of a spermatogenic cell squash preparation for the midpachytene marker H1t and for γH2AX shows that the majority of H1t-positive cells have fragmented γH2AX staining; only rare cells have a single sex body–like γH2AX-positive domain (bottom insets). In the control (top insets), cells with fragmented γH2AX staining (presumed to be zygotene) are H1t negative, whereas all H1t-positive spermatocytes have a single γH2AX-positive sex body (the small H1t-positive cells are round spermatids). (i) Quantitation of the whole nuclear γH2AX signal for rare pachytene spermatocytes with only the X and Y axes asynapsed and for pachytene spermatocytes with increasing levels of asynapsis shows that the amount of γH2AX does not increase in response to asynapsis (fitted blue regression line). The projected increase in γH2AX signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. Bars: (a–f) 10 μm; (g and h) 15 μm.
Mentions: Combined SYCP3, γH2AX, and ATR immunostaining indicated that in pachytene cells with limited asynapsis, there was a robust MSUC response covering the unsynapsed chromosome regions, including the unsynapsed X and Y axes (Fig. 3, a and b). However, with increasing asynapsis, the unsynapsed chromatin–located γH2AX staining became progressively more fragmented and weaker, and this was associated with increasingly focal ATR staining that was axially restricted without the spreading to adjacent chromatin that characterizes the MSUC response (Fig. 3, c–f). This fragmented γH2AX staining resembled that of meiotic DSB-associated γH2AX (Mahadevaiah et al., 2001). Amounts of γH2AX appeared to remain relatively constant in the face of increasing asynapsis; this was confirmed by quantitating the γH2AX signal (Fig. 3 i). Because the cells with fragmented and weak γH2AX staining had multiple regions of asynapsis and lacked a sex body or pseudo–sex body, we were concerned that they might have been zygotene spermatocytes, although there was often nonhomologous synapsis with the more extensive asynapsis (Fig. 3, e and f), which we consider a robust pachytene marker. For further confirmation, we stained testis tubule squashes for γH2AX and for the midpachytene marker histone H1t (Moens et al., 1997; Inselman et al., 2003), which appears during epithelial stage IV (Fig. S5, a–c; available at http://www.jcb.org/cgi/content/full/jcb.200710195/DC1). This confirmed that there were abundant H1t-positive Dnmt3l−/− spermatocytes that had fragmented γH2AX staining and no sex body; only rare midpachytene cells with a single sex body–like domain were seen (Fig. 3, g and h). Previously, Bourc'his and Bestor (2004) failed to detect H1t-positive Dnmt3l−/− spermatocytes with the same antibody, but they used spread cell preparations in which we have found that the weak stage IV H1t staining is not apparent.

Bottom Line: In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response.We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs.Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11- mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

Show MeSH
Related in: MedlinePlus